Peroxisome Proliferator-Activated Receptors



The Peroxisome Proliferator-Activated Receptors (PPAR) α, γ, and δ are members of the nuclear receptor family. Since their discovery in the early 90s, it has become clear that the PPARs are essential modulators of external stimuli, acting as transcription factors to regulate mammalian metabolism, cellular differentiation, and tumorigenesis. The PPARs are the targets of numerous pharmaceutical drugs aimed at treating hypolipidemia and diabetes among other diseases.

Biological Role
Transcription of individual genes in eukaryotic cells is controlled very precisely at a number of different levels. One key level is the binding of specific DNA binding transcriptional factors such as nuclear receptors, to facilitate RNA polymerase function. Unliganded PPARs form a heterodimer with retinoid X receptor (RXR), specifically RXRα. This heterodimer binds to the Peroxisome Proliferator Response Element (PPRE), a specific DNA sequence present in the promoter region of PPAR-regulated genes. Also associated with this unliganded heterodimer is a co-repressor complex which possesses histone deacetylation activity. This results in a tight chromatin structure, preventing gene transcription. This co-repressor complex is released upon ligand binding (typical ligands include lipids and eicosanoids), allowing various co-activators and co-activator-associated proteins to be recruited. These protein complexes facilitate chromatin remodeling and DNA unwinding along with linkage to RNA polymerase II machinery, necessary steps for transcription. The genes transcribed upon activation are insulin responsive genes involved in the control of glucose production, transport and utilization. This makes agonists of PPAR insulin sensitizers. Some PPAR related co-activators include CBP (Histone Acetylation), SRC-1,2,3 (Chromatin Acetylation), PGC-1 (Recruit HAT activities), PRIC-285,320 (Chromatin Remodeling via Helicase activity) and PIMT (RNA Capping via methyltransferase activity).

PPARs regulate diverse biological processes varying from lipid and carbohydrate metabolism to inflammation and wound healing. While PPARα is the major regulator of fatty acid oxidation and uptake in the liver, PPARγ is expressed at extremely high levels in adipose tissue, macrophages, and the large intestine, and controls lipid  adipogenesis and energy conversion. PPARδ is expressed in most tissues and plays diverse roles involved in metabolism and wound healing. These nuclear receptors are of critical importance to the body as exemplified by PPARα knockdown mice suffering from a variety of metabolic defects including hypothermia, elevated plasma free fatty acid levels, and hypoglycemia, potentially leading to death.

Natural Ligands
PPAR gamma binds polyunsaturated fatty acids like linoleic acid, linolenic acid, and eicosapentaenoic acid at affinities that are in line with serum levels found in the blood. PPARα binds a variety of saturated and unsaturated fatty acids including palmitic acid, oleic acid, linoleic acid, and arachidonic acid. PPARδs ligand selectivity is intermediate between that of the other isotypes and is activated by palmitic acid and a number of eicosanoids.

PPAR Structure


Ligand Binding Domain
The structures of the PPARs are very similar over each isotype. All PPAR isotypes have a ligand binding domain (LBD). The LBD, which is located in the C-terminal half of the receptor, is composed of 13 α-helices and a four-stranded ß-sheet. The ligand binding pocket (2f4b) is Y-shaped and consists of an entrance and two pockets, Arm I and Arm II, along with a "charge-clamp". The ligand binding pocket of PPARs is quite large (about 1400 cubic angstroms) in comparison to that of other nuclear receptors which allows the PPARs to interact with numerous structurally distinct ligands. . Within Arm I, four polar resides are conserved over all PPAR isotypes, namely Ser280, Tyr314, His440, and Tyr464 in the case of PPARα. These residues are part of a hydrogen bonding network that interacts with the carboxylate group of fatty acids and other ligands upon binding. The ligand-dependent activation domain (AF-2) helix H12 (1kkq), whose function is to generate the receptors’ co-activator binding pocket, is located at the C-terminal end of the LBD. The conserved hydrogen bonding network in Arm I also helps hold the AF2-helix in the active conformation, promoting co-activator binding. Arm II is highly hydrophobic and is thus ideal for binding the hydrophobic tail of fatty acids via Van der Waals interactions.

Despite over 80% of the ligand binding cavity residues being conserved over all PPAR isotypes, it is the remaining 20% that creates the ligand specificity seen between isotypes. A few examples illustrate this point. In PPARδ, the cavity is significantly narrower adjacent to the AF-2 helix and Arm I. This prevents PPARδ from being able to accommode large headed TZDs and L-tyrosine based agonsists. In the case of PPARα, PPARα does not bind ligands with large carboxylate head groups because of  Tyrosine 314 as compared to PPARγ which has a smaller equivalent residue in His323. Or in the case of binding some benzenesulfonamide derivatives, the pi stacking of Phe363 and the aromatic moiety (2g0g) in the case of PPARγ is lost in PPARα (Ile354) and PPARδ(Ile 363)

AF-2 Domain: Structure and Function
As briefly mentioned before, the AF-2 domain is essential for ligand binding and PPAR (2prg) function. Upon ligand binding, helix H12 of AF-2 closes on the ligand-binding site, reducing conformational flexibility of the LBD and assuming a structure that is ideal for co-activator binding. Using Molecular Dynamic simulations, it has been determined that residues Glu324, Arg397, Arg443, and Tyr 477 (in PPARγ) are involved in a hydrogen bond network that stabilizes the AF-2 helix in the active conformation upon ligand binding.

Co-Activator & Co-Repressor Binding
The transcriptional activity of PPAR is regulated by its interaction with co-activators like SRC-1 or CBP and co-repressors like SMRT. Co-activators like CBP contain a conserved LXXLL motif where X is any amino acid, and use this to bind a hydrophobic pocket on the receptor surface formed by the stabilized AF-2 helix H12. In the case of the PPARγ/rosiglitazone/SRC-1 complex, the LXXLL motif helix of SRC-1 forms hydrophobic interactions with Leu468 and Leu318 of the LBD and hydrogen bonds between Glu471 and Lys301 and the co-activator backbone. These charged residues are conserved across PPAR isotypes and form the “charge clamp,” an essential component for co-activator stabilization in the PPAR LBD.

When PPAR is bound to a co-repressor, the hydrogen bond between Tyr 464 in PPAR alpha in AF-2 and other AF-2 stabilizing helices is destroyed, preventing the AF-2 H12 helix from occupying its active state. This in turn eliminates the charge clamp between PPAR and a prospective co-activator. Notice the position of H12 when bound to a co-activator.

Formation of Heterodimer with RXR
The interface of PPAR and RXR is composed of an intricate network of hydrophobic and <scene name='Peroxisome_Proliferator-Activated_Receptors/Dimer_interface_polar/2'>polar interactions which show remarkable complementarity. For the PPARγ-RXRα dimer the dimmer interface interactions are particularly extensive.

DNA Binding Domain Structure
PPARs also contain a DNA binding domain (DBD) The <scene name='Peroxisome_Proliferator-Activated_Receptors/Zinc_fingers/1'>DBD consists of two zinc fingers (3dzy), one on PPAR and one on RXR, that bind PPREs of PPAR-responsive genes. The consensus sequence of PPREs is AGGTCA and has been found in a number of PPAR inducible genes like acyl-CoA oxidase and adipocyte fatty acid-binding protein. Chandre et al. have demonstrated that the DNA PPRE allosterically contributes to its own binding via a <scene name='Peroxisome_Proliferator-Activated_Receptors/Dbd_hbonds/1'>head-to-tail interaction between the PPAR DBD and RXR DBD using residues Gln206 and Arg209 on RXRα and Asn160 on PPARγ.

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Binding of Synthetic Agonists and Medical Implications
<applet load=" 3dzy2.pdb" size="450" color="white" frame="true" spin="on" Scene ="Peroxisome_Proliferator-Activated_Receptors/Ppar_opening4/2" caption="Crystal Structure of PPARγ bound to Rosiglitizone, RXRα and PPRE DNA Sequence, 3dzy" align="right"/> A number of synthetic agonists have been developed to bind to <scene name='Peroxisome_Proliferator-Activated_Receptors/Ppar_opening4/2'>PPAR to fight metabolic diseases like diabetes. These agonists include troglitazone (Rezulin), pioglitazone (Actos), and rosiglitazone (Avandia). These agonists function in a similar fashion, by binding to the active site of PPARγ, activating the receptor. Rosiglitazone occupies roughly 40% of the LBD. It assumes a U-shaped conformation with the TZD head group forming a <scene name='Peroxisome_Proliferator-Activated_Receptors/Rosiglitazone_binding/3'>number of interactions that stabilize the agonist. Rosiglitazone forms hydrogen bond interactions with H323 and H449 and its TZD group, the sulfur atom of the TZD occupies a hydrophobic pocket formed by Phe363, Glu286, Phe282, Leu330, Ile326 and Leu469, and the central benzene ring occupies a pocket formed by Cys285 and Met364. Despite their structural similarities, each member of the PPAR family is localized to certain parts of the body. Location of receptor partially determines their function in the body and also the different roles they can play in medicine as drug targets. PPARγ is responsible for lipid metabolism and cellular energy homeostasis. It binds genes that transcribe proteins which act as fatty acid transporters, are critical in insulin signaling and glucose transport, catalyze glycerol synthesis from triglycerides, and catabolize lipids. This makes PPARγ an ideal target to treat Diabetes. Also, recent research has indicated that some PPAR agonists like Rosiglitazone can induce apoptosis of macrophages and would thus serve as excellent anti-inflammatory targets. PPARα has been shown to play a critical role in the regulation of uptake and oxidation of fatty acids. This makes PPARα an excellent target for Atherosclerosis drugs which aim to reduce LDL cholesterol and increase HDL cholesterol, the two most common traits of atherosclerosis. The fibrates are a class of amphipathic carboxylic acids that are PPARα agonists used to treat hypercholesterolemia and hyperlipidemia along with the HMGR inhibitor statins. Some fibrates are Bezafibrate (Marketed by Roche as Bezalip) and Ciprofibrate (Modalim). PPARδ is broadly expressed across the human body and thus is suspected to play a role in a number of diseases. It has been implicated in disorders ranging from fertility problems to types of cancer. Perhaps the most important use of PPARδ agonists will be in treating central nervous system (CNS) diseases as PPARδ has been implicated in neuron myelinogenesis and neuronal signaling as well as lipid metabolism in the CNS.

Most drugs target the PPARγ LBD, as ligands that bind to RXRα are likely to inadvertently act on other RXRα complexes, resulting in unexpected side effects. Sales of Avandia, marketed by GlaxoSmithKline peaked at $2.5 billion in 2006 but have since dipped dramatically due to health concerns. In response to the health concerns, sales of Actos, marketed by Takeda, have grown to block buster status.

Additional 3D Structures of PPAR
Update June 2011

PPARγ Structures
2zk0, 2zk1, 2zk2, 2zk3, 2zk4, 2zk5, 2zk6 – hPPARγ LBD + ligand - human

2prg, 1fm6 – hPPARγ LBD + Rosiglitazone + SRC-1

3prg, 2qmv – hPPARγ LBD

4prg – hPPARγ LBD + 2,4-thiazolidinedione deriveative

1fm9 – hPPARγ LBD + GI262570, Farglitazar + SRC-1

1wm0 – hPPARγ LBD + 2-BABA + GRIP-1

3ho0, 3hod – hPPARγ LBD + aryloxy-3phenylpropanoic acid

1k74 – hPPARγ LBD + retinoicic acid receptor + inhibitor

3et0 - hPPARγ LBD + propionic acid moiety

1knu – hPPARγ LBD + Carbazole analogue

1i7i – hPPARγ LBD + AZ242

2fvj – hPPARγ LBD + Isoquinoline derivative + SRC-1

1nyx – hPPARγ LBD + Ragalitazar

1rdt – hPPARγ LBD + GI262570, Fraglitazar + CBP

1zgy – hPPARγ LBD + Rosaglitazone + SHP

2f4b, 1zeo, 2ath, 2hwq, 2hwr, 2i4j, 2q59, 2q8s, 3b3k, 3bc5, 3cds, 3g9e, 3gbk, 3gz9, 3ia6, 3kdt – hPPARγ LBD+ agonists

2g0h, 2g0g, 2i4p, 2i4z, 2q5g, 2q5p, 2q5s, 2q61, 2q6r, 3cdp, 3d6d - hPPARγ LBD+ partial agonists

3lmp – hPPARγ LBD + a cercosporamide derivative modulator

2gtk - hPPARγ LBD+ SRC-1 decamer

2om9 - hPPARγ LBD + ajulemic acid

2q6s - hPPARγ LBD + benzoic acid derivative

3osi, 3osw - hPPARγ LBD + bisphenol derivative

2p4y, 3adt, 3adu, 3adw, 3et3, 2hfp - hPPARγ LBD+ indole modulator

2pob - hPPARγ LBD + fraglitazar analogue

2vsr, 2vst, 2vv0, 2vv1, 2vv2, 2vv3, 2vv4 - hPPARγ LBD + fatty acid activator

2zvt - hPPARγ LBD + prostaglandin derivative

3ads, 3adx - hPPARγ LBD + indomethacin

3adv - hPPARγ LBD + serotonin

3cs8 - hPPARγ LBD + PGC-1A

3cwd - hPPARγ LBD + SRC1-2

3fur, 3kmg - hPPARγ LBD + SRC-1+ modulator

3k8s - hPPARγ LBD + antidiabetic agent

3h0a - hPPARγ LBD + SRC 1 + retinoic acid receptor α + retinoic acid + partial agonist

PPARα Structures
1k7l – hPPARα LBD + G2409544 + SRC-1

3e94 – hPPARα LBD + tributyltin

1i7g – hPPARα LBD + AZ242

1kkq – hPPARα LBD + GW6471 Antagonist + SMRT

2npa - hPPARα LBD+ propanoic acid derivative + SRC-1

2p54 - hPPARα LBD + SRC-1

2rew - hPPARα LBD + azetidinone derivative activator

2znn, 2zno, 2znp, 2znq, 3kdu - hPPARα LBD+ agonist

3fei, 3fej, 3g8i - hPPARα LBD+ agonist + SRC-1

3et1 - hPPARα LBD + SRC-1 + indole derivative

PPARδ Structures
2baw, 2b50, 2awh – hPPARδ + Vaccenic Acid

1gwx – hPPARδ LBD + GW2433

2gwx – hPPARδ LBD

3gwx –hPPARδ LBD + 5,8,11,14,17-Eicosapentaenoic Acid

1y0s – hPPARδ LBD + GW2331

3dy6 –hPPARδ LBD + anthranilic acid

3et2 – PPARδ + 3-[5-Methoxy-1-(4-methoxy-benzenesulfonyl)-1H-indol-3-yl]-propionic acid

2env – hPPARδ zinc finger domain

2j14, 2xyj, 2xyw, 2xyx, 3oz0 - hPPARδ LBD + agonist

3d5f - hPPARδ LBD +phenoxy derivative

Additional Resources

 * See: Regulation of Gene Expression For Additional Mechanisms of Gene Regulation
 * See: Pharmaceutical Drug Targets For Additional Information about Drug Targets for Related Diseases
 * See: Diabetes & Hypoglycemia For Additional Information about Diabetes & Hypoglycemia Related Information